Bispecific antibody effective to treat B-cell lymphoma and cell line

ABSTRACT

The invention provides bispecific antibodies with selective cytotoxicity against malignant B-cells. The bispecific antibodies bind to an effector cell antigen and to a 28/32 kDa heterodimeric protein on the surface of malignant B-cells. The invention also includes the monospecific components of the bispecific antibodies, humanized versions thereof, and humanized bispecific antibodies. The invention further provides therapeutic and diagnostic methods employing these antibodies.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. Ser. No. 07/859,583filed Mar. 27, 1992, now abandoned, which is incorporated by referencein its entirety for all purposes.

BACKGROUND OF THE INVENTION

Administration of monoclonal antibodies (MoAb) has shown promise as anew treatment modality for human malignancy. However, destruction ofmalignant cells by MoAb does not always occur, even after successfulbinding of the antibody to the target cell. A second approach toimmunotherapy of malignancy involves the manipulation of the cellularimmune system. Lymphokines, such as IL-2, can be used to activate bothNK cells and T cells isolated from the blood, spleen, or malignanttumors themselves. The anti-tumor effects of such cells have been welldocumented both in vitro and in vivo. Toxicity of therapy based on IL-2alone can be severe and may well limit the clinical utility of thistherapy.

Immunotherapy of malignancy that attempts to combine the specificity ofantibodies with the power of activated lymphocytes might be moreeffective and less toxic. One such approach is the use of bispecificantibodies to redirect activated T cell toxicity toward tumor cellsexpressing the target antigen (Ag.)

Various forms of bispecific antibodies have been produced. These includeBSIgG, which are IgG molecules comprising two distinct heavy chains andtwo distinct light chains that are secreted by so-called "hybridhybridomas", and heteroantibody conjugates produced by the chemicalconjugation of antibodies or antibody fragments of differentspecificities.

Several investigators have evaluated anti-CD3/anti-tumor bispecificantibody structures as immunotherapeutic agents. Such studies havereported in vitro cytolysis of renal cell carcinoma, melanoma, glioma,lymphoma, leukemia and cells expressing the multidrug-resistance-relatedglycoprotein. IL-2-activated human peripheral lymphocytes directed bycertain anti-CD3/anti-tumor-specific heteroantibody conjugates have alsobeen reported to prevent the growth of human cancer xenografts in nudemice. Studies in vitro, and in vivo in immunodeficient mice bearinghuman xenografts have reported that certain bispecific antibodies arecapable of blocking the growth of both tumor cells bearing certaintarget antigens and, to some extend, bystander tumor cells that are notrecognized by the therapeutic antibody.

The cell membranes of lymphocytes are uniquely constructed and determinesuch diverse cellular phenotypic characteristics as the suppressor,inducer, or cytolytic function of the cell, the state of activation orstage of differentiation of the cell, and whether the cell belongs to apopulation that is monoclonal or polyclonal. The vast majority ofcellular membrane antigens thus far described on malignant lymphocytesare represented on nonmalignant lymphocytes at some stage ofdifferentiation or activation.

From the foregoing, it is apparent that a need exists for therapeuticagents that are targeted to an antigen found predominantly orexclusively on malignant cells, and which are capable of inducing strongcytolytic activity against such cells. The present invention fulfillsthis and other needs.

SUMMARY OF THE INVENTION

The present invention is premised on the realization that a bispecificmonoclonal antibody which binds to malignant B-cell lymphomas and to Tcells can be formed which effectively binds only to malignant B-cellsand does not bind to normal B-cells.

Further, the present invention is premised on the realization that abispecific antibody can be formed from a cell line obtained fromperipherally diffuse large cell lymphoma to produce a monoclonalantibody that is specific only to malignant B-cells and that thismonoclonal antibody can be modified to form a bispecific antibody whichalso binds to killer T cells or NK cells.

The present invention is further premised on the realization that a cellline formed from a fusion of cell lines which produces an IgG antibodyspecific to the T cells or NK cells and a cell line which produces theIgG antibody specific to B-cell malignancies in turn produce a uniquebispecific antibody that effectively binds to both malignant B-cells andT cells or NK cells thereby effectuating the lysis or destruction of themalignant B-cells.

In the preferred embodiment the cell line is derived from the fusion ofa cell line producing an antibody specific to the CD3 antigen of the Tcell in combination with a cell line specific to a heterodimer on thecell membrane of the malignant B-cells as explained further below.

In a further aspect, the invention provides the 1D10 antibody, which isspecific for the 28/32 kDa heterodimeric protein on the surface ofmalignant B-cells.

The invention further provides a humanized version of the 1D10 antibody.The humanized antibody comprises a humanized heavy chain and a humanizedlight chain. The humanized light chain comprises three complementaritydetermining regions (CDR1, CDR2 and CDR3) having amino acid sequencesfrom the corresponding complementarity determining regions of the 1D10immunoglobulin light chain, and a variable region framework from a humankappa light chain variable region framework sequence except in at leastone position selected from a first group consisting of L48, L49, L69,and L70 wherein the amino acid position is occupied by the same aminoacid present in the equivalent position of the 1D10 immunoglobulin lightchain variable region framework. The humanized heavy chain comprisingthree complementarity determining regions (CDR1, CDR2 and CDR3) havingamino acid sequences from the corresponding complementarity determiningregions of 1D10 immunoglobulin heavy chain, and a variable regionframework from a human heavy chain variable region framework sequenceexcept in at least one position selected from a second group consistingof H27, H29, H30, H37, H67, H71, H78 and H83, wherein the amino acidposition is occupied by the same amino acid present in the equivalentposition of the mouse 1D10 immunoglobulin heavy chain variable regionframework. The humanized antibody specifically binds the 28/32 kDaheterodimeric protein cells with a binding affinity having a lower limitof about 10⁷ M⁻¹ and an upper limit of about five-times the bindingaffinity of the 1D10 immunoglobulin. Preferably, the humanized lightchain variable region framework is from the R3.5H5G antibody. In thiscase, position L43 can be substituted with the amino acid present in theequivalent position of a human kappa subgroup I consensus sequence.Preferably, the humanized heavy chain is from the heavy chain regionvariable framework of the IC4 antibody. In this case, position H73 canbe substituted by the same amino acid present in the equivalent positionof a human immunoglobulin subgroup II or IV consensus sequence.

In a further aspect, the invention provides humanized antibodiesspecific for the CD3 antigen. The antibodies comprise humanized heavyand light chains. The humanized light chain comprises threecomplementarity determining regions (CDR1, CDR2 and CDR3) having aminoacid sequences from the corresponding complementarity determiningregions of the M291 immunoglobulin light chain, and a variable regionframework from a human kappa light chain variable region frameworksequence. The humanized heavy chain comprises three complementaritydetermining regions (CDR1, CDR2 and CDR3) having amino acid sequencesfrom the corresponding complementarity determining regions of M291immunoglobulin heavy chain, and a variable region framework from a humanheavy chain variable region framework sequence except in at least oneposition selected from a second group consisting of H30, H67, H68, H70,H72 and H74 wherein the amino acid position is occupied by the sameamino acid present in the equivalent position of the mouse M291immunoglobulin heavy chain variable region framework. The immunoglobulinspecifically binds to a CD3 antigen on the surface of T cells with abinding affinity having a lower limit of about 10⁷ M⁻¹ and an upperlimit of about five-times the binding affinity of the M291immunoglobulin. Preferably, the humanized light chain variable regionframework is from the light chain variable region framework of theHF2-1/17 antibody in subgroup I. Preferably, the humanized heavy chainregion framework is from the heavy chain region variable framework ofthe 21/28 antibody. In this case, position H44 can be substituted withthe same amino acid present in the equivalent position of a humanimmunoglobulin subgroup I consensus sequence.

In a further aspect, the invention provides humanized bispecificantibodies comprising a first binding fragment that specifically bindsto the CD3 antigen and a second binding fragment that specifically bindsto the 28/32 kDa heterodimeric antigen on the surface of the malignant Bcells. The first binding fragment comprises a humanized form of theheavy chain variable region of the M291 antibody and a humanized form ofthe light chain variable region of the M291 antibody. The second bindingfragment, which is linked to the first binding fragment, comprising: ahumanized form of the heavy chain variable region from the 1D10 antibodyand a humanized form of the light chain variable region from the 1D10antibody.

Preferably, the first and second binding fragments each furthercomprises a segment of a constant region fused to the respective heavychain variable regions, and the binding fragments are linked byassociation of the constant regions. For, example, the binding fragmentscan be Fab or Fab'. When both binding fragments are Fab', the bispecificantibody is a F(ab')₂. Optionally, the first and second bindingfragments further comprise first and second leucine zippers fused to therespective constant regions.

The invention further provides pharmaceutical compositions comprisingthe antibodies described above. Also provided are methods of treatingpatients suffering from malignant B-cells employing a therapeuticallyeffective amount of bispecific antibody as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting lysis of malignant B-cells by the bispecificantibody of the present invention;

FIG. 2 is a graph depicting lysis of Raji cells caused by differentconcentrations of the antibody of the present invention;

FIG. 3 is a graph depicting lysis of KH cells over a period of time bythe bispecific antibody of the present invention also depicting acomparative study.

FIG. 4. Amino acid sequences of the light chain (A) (SEQ ID NOS. 1 and2) and the heavy chain (B) (SEQ ID NOS. 3 and 4) variable regions of thehumanized 1D10 antibody (upper lines) and mouse 1D10 antibody (lowerlines), not including the signal sequences. The three CDRs in each chainare underlined Residues in the human framework that have been replacedwith mouse amino acids or consensus human amino acids are doublyunderlined. Amino acid sequences of the complete light chain and theheavy chain of the humanized 1D10 are showed in (C) (SEQ ID NO. 5) and(E) (SEQ ID NO. 7) , respectively. The V_(L) domain consists of residues1-107, and the C_(K) 108-214. The V_(H) domain consists of residues1-116, the C_(H) 1 117-214, the hinge 215-229, the C_(H) 2 230-339, andthe C_(H) 3 domain 340-446. Amino acid sequence of the Fd-Jun in thehumanized F(ab'-zipper)₂ of 1D10 is shown in (D) (SEQ ID NO. 6). TheV_(H) domain consists of residues 1-116, the C_(H) 1 domain 117-214, themodified hinge 215-234, and the Fos leucine zipper 235-273.

FIG. 5. Amino acid sequences of the light chain (A) (SEQ ID NOS. 8 and9) and the heavy chain (B) (SEQ ID NOS. 10 and 11) variable regions ofthe humanized M291 antibody (upper lines) and mouse M291 antibody (lowerlines), not including the signal sequences. The three CDRs in each chainare underlined. Residues in the human framework that have been replacedwith mouse amino acids or consensus human amino acids are doublyunderlined. Amino acid sequences of the complete light chain of thehumanized M291 are showed in (C) (SEQ ID NO. 12). The V_(L) domainconsists of residues 1-106, and the human C_(K) domain 107-213. Aminoacid sequence of the Fd-Fos in the humanized F(ab'-zipper)2 of M291 isshown in (D) (SEQ ID NO. 13). The V_(H) domain consists of residues1-120, the C_(H) 1 domain 121-218, the modified hinge 219-238, and theFos leucine zipper 239-279.

FIG. 6. Construction of the plasmid pHu1D10.IgG1.rG.dE used for theexpression of the humanized 1D10 IgG1.

FIG. 7. (A). Displacement assay to compare the relative affinity ofhumanized 1D10 and murine 1D10 for the antigen. Subsaturation amounts ofmurine 1D10-IgG2a-FITC on Raji cells were displaced by increasingamounts of murine 1D10-IgG2a or humanized 1D10-IgG1. Raji cells wereresuspended in complete media at 2.5×10⁶ /ml. Dilutions of the test(humanized 1D10-IgG1) or control (murine 1D10-IgG2a) antibody were addedand incubated at 4° C. for 1 hour. A fixed, subsaturation amount ofmurine 1D10-IgG2a-FITC was added, and the cells were incubated at 4° C.for 1 hour, washed, and resuspended in 1% paraformaldehyde. The cellswere then analyzed using flow cytometry. Values expressed in %inhibition of fluorescence intensity compared to no competitive antibodycontrol. (B). Scatchard plot analysis of the binding of ¹²⁵ I-labeledhumanized 1D10-IgG1 to Raji cells. Scatchard analysis was made bybinding dilutions of labeled antibody to 4×10⁵ Raji cells in 0.2 ml for90 min at 0° C. The cells were washed in binding buffer (2% horse serumin PBS containing 0.1% sodium azide) and counted. Nonspecific bindingwas determined by inhibiting the specific binding with an excess ofnonlabeled humanized 1D10-IgG1. The apparent Ka and the number ofbinding sites were calculated from the slope and the X axis intercept,respectively, of the Scatchard plot.

FIG. 8. (A). Antibody-dependent cell-mediated cytotoxicity (ADCC)capability by various 1D10 isotypes. ⁵¹ Cr-labeled Raji human lymphomacells were used as targets for (▴) murine 1D10-IgG1, () murine1D10-IgG2a, or (▪) humanized 1D10-IgG1 and human peripheral mononuclearas effector cells. The effector:target ratio was 40:1. Spontaneousrelease was less than 20% of total release. Bars represent SEM. (B).Complement-mediated cytotoxicity by various 1D10 isotypes. ⁵¹ Cr-labeledRaji human lymphoma cells were used as targets for (▴) murine 1D10-IgG1,() murine 1D10-IgG2a, or (▪) humanized 1D10-IgG1 and human sera from anormal subject as complement. Spontaneous release was less than 20% oftotal release. Bars represent SEM.

FIG. 9. Schematic diagrams of the plasmids pHu1D10-Jun.rG.dE andpHuM291-Fos.rG.dE for the expression of Hu1D10-Jun and HuM291-FosF(ab'-zipper)₂. The constructions of these two plasmids were similar tothat of pHu1D10.IgG1 in FIG. 6 except for the replacement of the C_(H) 2and C_(H) 3 exons by the leucine zipper sequences Jun and Fos. Thepolyadenylation signal for the Fd-zipper transcript is from the 3'noncoding sequence of mouse IgG2a gene (see Kostelny et al., J. Immunol.148, 1547 (1992)).

FIG. 10. (A). The sequence of the modified human IgG1 hinge used in thehinge-zipper fusion. Two residues Lys-Cys (underlined) were inserted inthe modified hinge. The fist Cys in this modified hinge forms disulfidebond with the light chain, and the last three Cys residues forminter-heavy chain disulfides. For comparison, hinge sequences of thehuman IgG1 (B) and the mouse IgG2a (C) are also shown. All three Cysresidues in the mouse IgG2a hinge are used for inter-heavy chaindisulfides. After the insertion of Lys-Cys, the modified hinge and themouse IgG2a hinge have extensive sequence homology near theCOOH-terminus.

FIG. 11. (A). Displacement assay to compare the relative affinity ofHuM291-Fos and M291 for their antigen. Subsaturation amounts of murineM291-FITC on human T cells were displaced by increasing amounts ofmurine M291 or HuM291-Fos. T cells were resuspended in complete media at2.5×10⁶ /ml. Dilutions of the test (HuM291-Fos) or control (murine M291)antibody were added and incubated at 4° C. for 1 hour. A fixed,subsaturation amount of murine M291-FITC was added, and the cells wereincubated at 4° C. for 1 hour, washed, and resuspended in 1%paraformaldehyde. The cells were then analyzed using flow cytometry.Values expressed in % inhibition of fluorescence intensity compared tono competitive antibody control. (B). Scatchard plot analysis of thebinding of ¹²⁵ I-labeled HuM291-Fos to activated human T cells.Scatchard analysis was made by binding dilutions of labeled antibody to4×10⁵ T cells in 0.2 ml for 90 min at 0° C. The cells were washed inbinding buffer (2% horse serum in PBS containing 0.1% sodium azide) andcounted. Nonspecific binding was determined by inhibiting the specificbinding with an excess of nonlabeled HuM291-Fos. The apparent K_(a) andthe number of binding sites were calculated from the slope and the Xaxis intercept, respectively, of the Scatchard plot.

FIG. 12. Bispecific antibody induced T cell mediated lysis of 1D10positive cells. T cells in human PBL were activated by anti-CD3 antibodyOKT3 and expanded by culturing them in IL-2. Target cells were labeledwith ⁵¹ Cr and washed. T cells and labeled target cells at effector:target ratio of 25:1 were plated in V bottom microtiter plates.Antibodies at desired concentration were added. Antibodies used were:Hu1D10-Jun, HuM291-Fos, the mouse bispecific IgG 1DT3-D, and thehumanized bispecific F(ab'-zipper)₂ Hu1D10-Jun×HuM291-Fos. Plates wereincubated at 37° C. for 4 hours, centrifuged, and target cell lysis wasmeasured by determining the amount of ⁵¹ Cr released. Percentages ofspecific release in this cytotoxicity assay were calculated as: {Countsreleased by antibody minus counts released without addedantibody}/{Counts released by 0.1% SDS minus counts released withoutadded antibody}×100.

DEFINITIONS

The term "substantial identity" or "substantial homology" means that twopeptide sequences, when optimally aligned, such as by the programs GAPor BESTFIT using default gap weights, share at least 65 percent sequenceidentity, preferably at least 80 or 90 percent sequence identity, morepreferably at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions which are notidentical differ by conservative amino acid substitutions.

For purposes of classifying amino acids substitutions as conservative ornonconservative, amino acids are grouped as follows: Group I(hydrophobic sidechains): norleucine, met, ala, val, leu, ile; Group II(neutral hydrophilic side chains): cys, ser, thr; Group III (acidic sidechains): asp, glu; Group IV (basic side chains): asn, gln, his, lys,arg; Group V (residues influencing chain orientation): gly, pro; andGroup VI (aromatic side chains): trp, tyr, phe. Conservativesubstitutions involve substitutions between amino acids in the sameclass. Non-conservative substitutions constitute exchanging a member ofone of these classes for a member of another.

Amino acids from the variable regions of the mature heavy and lightchains of immunoglobulins are designated Hx and Lx respectively, where xis a number designating the position of an amino acids according to thescheme of Kabat, Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md., 1987 and 1991). Kabatlists many amino acid sequences for antibodies for each subclass, andlists the most commonly occurring amino acid for each residue positionin that subclass. Kabat uses a method for assigning a residue number toeach amino acid in a listed sequence, and this method for assigningresidue numbers has become standard in the field. Kabat's scheme isextendible to other antibodies not included in his compendium byaligning the antibody in question with one of the consensus sequences inKabat. The use of the Kabat numbering system readily identifies aminoacids at equivalent positions in different antibodies. For example, anamino acid at the L50 position of a human antibody occupies theequivalent position to an amino acid position L50 of a mouse antibody.

From N-terminal to C-terminal, both light and heavy chains comprise thedomains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of aminoacids to each domain is in accordance with the definitions of Kabat(1987) and (1991), supra, or Chothia & Lesk, J. Mol. Biol. 196:901-917(1987); Chothia et al., Nature 342:878-883 (1989).

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one "light" (about 25 kDa) and one "heavy" chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Thevariable regions of each light/heavy chain pair form the antibodybinding site. Thus, an intact antibody has two binding sites.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya "J" region of about 12 or more amino acids, with the heavy chain alsoincluding a "D" region of about 10 more amino acids. (See generally,Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989),Ch. 7 (incorporated by reference in its entirety for all purposes).

The term epitope includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor. Epitopic determinantsusually consist of chemically active surface groupings of molecules suchas amino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics.

The term patient includes human and veterinary subjects.

DETAILED DESCRIPTION

The present invention provides bispecific antibodies, which are specificto both effector cells (T cells or natural killer cells) and to a 28/32kDa heterodimeric antigen present on the surface of malignant B-cells.The present invention further provides hybridomas and other cells linesproducing the claimed antibodies.

The 28/32 kDa antigen is found predominantly on the surface of malignantB lymphocytes and is not expressed on resting lymphocytes or B and Tcells activated in vitro by a variety of inductive stimuli. See Gingrichet al., Blood 75, 2375-2387 (1990). The antigen can be expressed whenlymphocytes undergo malignant transformation or, in some cases, whenthey are perturbed by the Epstein-Barr virus (EBV). Normal resting andstimulated lymphocytes do not express the antigen. The antigen is alsoabsent on hemopoietic stem cells. Although the scientific basis for the28/32 kDa antigen being expressed predominantly or exclusively onmalignant B-cells is not critical to the practice of the invention, itis believed that the antigen may represent an aberrantpost-translational processing variant of the HLA-Dr antigen.

To produce the antibodies specific to malignant B-cells, a lymphoma cellline derived from a patient with peripheralizing diffuse large celllymphoma labeled HO-85 was grown in suspension culture RPMI 1640 with10% fetal calf serum with a doubling time of approximately 24 hours. Thecell line is CD20, mu, delta (weakly), kappa, HLA Class I and II antigenpositive. It does not react with monoclonal antibodies detecting CALLA,T cell, myeloid or monocytic cell antigens. The cells react with theSFR7, DR7 and B7/21 monoclonal antibodies indicating that they expressDR7 and DP antigens respectively.

Female BALB/c mice, age 6-10 weeks, were given 4 to 6 intraperitonealinoculations at two week intervals with 5×10⁶ cells from the human largecell lymphoma line as described above. The animals were killed five daysafter last inoculation and the spleen cells were fused with thenonsecretory murine myeloma cell line N-1. Hybridomas were selected inhypoxanthine-aminopterin-thymidine (HAT) medium after being plated in 96well cell culture trays. After 10 days, 25 microliter aliquots weretaken from each well for determination of malignant B-cell (anti-HO-85)antibody binding activity.

Malignant B-cell (anti-HO-85) antibody binding activity was determinedby a whole cell, indirect radio immune assay using fresh HO-85 cells astargets. The identical assay was done using as targets RAJI (ATCCCCL86), MOLT-3 (ATCC CRL1582), HL-60 (ATCC CCL240) and fresh peripheralblood mononuclear cells. Wells that showed binding activity greater than5 times that of tissue culture medium alone to HO-85 and Raji but werenot reactive with MOLT-3, HL-60 and peripheral blood mononuclear cellswere harvested.

Cells meeting the above criteria were found to produce an antibodyreferred to as 1D10 and were subsequently cloned by limiting dilution.The hybridoma grows well in vitro and ascites of pristine-primed BALB/cmice.

The portion of the malignant B-cells to which 1D10 binds is aheterodimeric polypeptide which contains two proteins with a molecularweight of the alpha and beta chains being 32 kDa and 28 kDarespectively. The proteins can be obtained by solubilizing malignantB-cells such as Raji cells with detergent. Molecular weightdetermination is made by using iodinated cells and single dimensionSDS-PAGE analysis of the MoAb6-antigen precipitate. The formation of the1D10 antibody is discussed by Gingrich et al., Blood 75, 2375-2387(1990). Other antibodies having the same or similar binding specificityto 1D10 are screened by competition binding with 1D10 to the 28/32 kDaheterodimeric antigen. Numerous types of competitive binding assays areknown, for example: solid phase direct or indirect radioimmunoassay(RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwichcompetition assay (see Stahli et al., Methods in Enzymology 9, 242-253(1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J.Immunol. 137, 3614-3619 (1986)); solid phase direct labeled assay, solidphase direct labeled sandwich assay (see Harlow & Lane, "Antibodies, ALaboratory Manual," Cold Spring Harbor Press (1988)); solid phase directlabel RIA using I-125 label (see Morel et al., Molec. Immunol. 25, 7-15(1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology176, 546-552 (1990)); and direct labeled RIA (Moldenhauer et al., Scand.J. Immunol. 32, 77-82 (1990)). Typically, such an assay involves the useof cells bearing the 28/32 kDa antigen, an unlabelled testimmunoglobulin and a labelled reference immunoglobulin (1D10).Competitive inhibition is measured by determining the amount of labelbound to the cells in the presence of the test immunoglobulin. Usuallythe test immunoglobulin is present in excess. Antibodies identified bycompetition assay (competing antibodies) include antibodies binding tothe same epitope as the reference antibody and antibodies binding to anadjacent epitope sufficiently proximal to the epitope bound by thereference antibody for steric hindrance to occur.

The second component for the bispecific antibodies of the invention isan antibody having specificity for an antigen on the surface of T-cellsor NK cells. Human T-cell antigens likely to be suitable include CD3,CD2, CD28, CD44, C69, A13 and G1. Suitable antigens on natural killercells include FC Gamma receptors (3G8, B73.1, LEUL1, VEP13, and AT10).Human T-cell antigens that are probably unsuitable include MHC Class I,CD4, CD8, CD18 and CD71.

Cell lines producing IgG specific to the effector cell antigensdescribed above are commercially available or can be produced de novo(see Example 3). The OKT3 cell (ATCC CRL 8001) is a suitable source ofantibodies for the CD3 antigen. Other antibodies to the CD3 antigeninclude WT31, WT32, anti-leu-4, UCHT-1, SPV-3TA and SPV-T3B. The CD3site is preferred because of its presence in all T cells.

The antibodies of the present invention can be produced by a cell lineformed by the fusion of a first component cell line producing antibodiesspecific for the 28/32 kDa heterodimeric antigen with a second cell linewhich produces an antibody specific for either T cells or natural killercells. For example, the hybridoma producing 1D10 was fused with OKT3 asfollows.

The OKT3 hybridoma cell line was selected by growing OKT3 cellssequentially in media containing 0.13 mM 8-azaguanine, then 1.0 mMouabain. Hybrid-hybridomas were produced by fusion (using 38%polyethylene glycol) of 10⁶ HAT resistant, ouabain sensitive,1D10-secreting hybridomas with 10⁶ HAT sensitive, ouabain resistantOKT3-secreting hybridomas.

Fused cells were plated in HAT-ouabain media to select forhybrid-hybridomas. The HAT in this media prevented the growth of unfusedOKT3 cells and the ouabain prevented the growth of unfused 1D10 cells.Thus, only hybrid-hybridomas containing genetic material from bothparental hybridomas survived. Twelve hybrid-hybridomas were isolatedusing this technique.

Cell lines secreting bispecific antibodies can be identified by athree-step screening procedure. For example, in analysis of hybridomasformed from fusion of 1D10 and OKT3, an initial screen was performed inwhich hybrid-hybridoma supernatant was added to ELISA plates coated withgoat anti-mouse IgG1 antibody. After washing, alkaline phosphataselabeled goat anti-mouse IgG2a was added. Reactivity indicated thehybrid-hybridoma supernatant contained single antibody molecules withboth IgG1 and IgG2a heavy chains.

An indirect immunofluorescent assay was used as a second screen for allsamples that were positive on ELISA. In this second screen,hybrid-hybridoma supernatant was added separately to HO-85 (1D10reactive) and Jurkat (OKT3 reactive) cells. Goat anti-mouse IgG-FITC wasadded after washing to detect the presence of bound antibody. All twelvehybrid-hybridomas secreted antibody which was capable of binding to bothHO-85 and Jurkat cells. One of these hybrid-hybridomas was selected forfurther study. It was subcloned by limiting dilution ×2, and designated1DT3-D. This cell line was deposited on Mar. 24, 1992 under the BudapestTreat at the American Type Culture collection, 12301 Parklawn Drive,Rockville, Md. 20852 and assigned the number ATCC HB 10993.

1DT3-D was cultured in vitro in HB 101 media supplemented with 100 μgL-glutamine and 100 U/ml penicillin-streptomycin. These cells weretransferred to a Mini Flo-path Bioreactor hollow fiber apparatus.Antibody obtained from spent media was fractionated by HPLC cationexchange using a gradient of 0.18 to 0.5M NaCl. The peak containingbispecific reactivity, as demonstrated by the above assays, wasisolated, dialyzed against phosphate buffered saline, concentrated andused in further studies.

The bispecific antibody formed by fusion of 1D10 and OKT3 is a mousederived monoclonal. Humanized versions of this antibody and otherbispecific antibodies of the invention can also be employed as discussedin more detail below.

Humanized Antibodies

The invention further provides humanized immunoglobulins (orantibodies). Some humanized antibodies are specific for the T-cellantigen CD3. Other humanized antibodies are specific for the 28/32 kDaheterodimer on malignant B-cells. These humanized antibodies are usefulas therapeutic and diagnostic reagents in their own right or can becombined to form a humanized bispecific antibody possessing both of thebinding specificities of its components. The humanized forms ofimmunoglobulins have variable framework region(s) substantially from ahuman immunoglobulin (termed an acceptor immunoglobulin) andcomplementarity determining regions substantially from a mouseimmunoglobulin (referred to as the donor immunoglobulin). The constantregion(s), if present, are also substantially from a humanimmunoglobulin. The humanized antibodies exhibit a specific bindingaffinity for their respective antigens of at least 10⁷, 10⁸, 10⁹, or10¹⁰ M⁻¹. Often the upper and lower limits of binding affinity of thehumanized antibodies are within a factor of three or five or ten of thatof the mouse antibody from which they were derived.

(1) Mouse Antibodies for Humanization

The starting material for production of humanized antibody specific forthe 28/32 kDa heterodimer is preferably the 1D10 mouse antibody,although other mouse antibodies, which compete with 1D10for binding tothe 28/32 kDa heterodimer can also be used. A suitable starting materialfor production of humanized antibody specific for CD3 is the M291antibody whose isolation is described in Example 3.

(2) Selection of Human Antibodies to Supply Framework Residues

The substitution of mouse CDRs into a human variable domain framework ismost likely to result in retention of their correct spatial orientationif the human variable domain framework adopts the same or similarconformation to the mouse variable framework from which the CDRsoriginated. This is achieved by obtaining the human variable domainsfrom human antibodies whose framework sequences exhibit a high degree ofsequence identity with the murine variable framework domains from whichthe CDRs were derived. The heavy and light chain variable frameworkregions can be derived from the same or different human antibodysequences. The human antibody sequences can be the sequences ofnaturally occurring human antibodies or can be consensus sequences ofseveral human antibodies.

Suitable human antibody sequences are identified by computer comparisonsof the amino acid sequences of the mouse variable regions with thesequences of known human antibodies. The comparison is performedseparately for heavy and light chains but the principles are similar foreach.

(3) Computer Modelling

The unnatural juxtaposition of murine CDR regions with human variableframework region can result in unnatural conformational restraints,which, unless corrected by substitution of certain amino acid residues,lead to loss of binding affinity. The selection of amino acid residuesfor substitution is determined, in part, by computer modelling. Computerhardware and software for producing three-dimensional images ofimmunoglobulin molecules are widely available. In general, molecularmodels are produced starting from solved structures for immunoglobulinchains or domains thereof. The chains to be modelled are compared foramino acid sequence similarity with chains or domains of solved threedimensional structures, and the chains or domains showing the greatestsequence similarity is/are selected as starting points for constructionof the molecular model. The solved starting structures are modified toallow for differences between the actual amino acids in theimmunoglobulin chains or domains being modelled, and those in thestarting structure. The modified structures are then assembled into acomposite immunoglobulin. Finally, the model is refined by energyminimization and by verifying that all atoms are within appropriatedistances from one another and that bond lengths and angles are withinchemically acceptable limits.

(4) Substitution of Amino Acid Residues

As noted supra, the humanized antibodies of the invention comprisevariable framework region(s) substantially from a human immunoglobulinand complementarity determining regions substantially from a mouseimmunoglobulin (e.g., 1D10 or M291). Having identified thecomplementarity determining regions of mouse antibodies and appropriatehuman acceptor immunoglobulins, the next step is to determine which, ifany, residues from these components should be substituted to optimizethe properties of the resulting humanized antibody. In general,substitution of human amino acid residues with murine should beminimized, because introduction of murine residues increases the risk ofthe antibody eliciting a HAMA response in humans. Amino acids areselected for substitution based on their possible influence on CDRconformation and/or binding to antigen. Investigation of such possibleinfluences is by modelling, examination of the characteristics of theamino acids at particular locations, or empirical observation of theeffects of substitution or mutagenesis of particular amino acids.

When an amino acid differs between a mouse variable framework region andan equivalent human variable framework region, the human framework aminoacid should usually be substituted by the equivalent mouse amino acid ifit is reasonably expected that the amino acid:

(1) noncovalently contacts antigen directly, or

(2) is adjacent to a CDR region or otherwise interacts with a CDR region(e.g., is within about 4-6 Å of a CDR region).

Other candidates for substitution are acceptor human framework aminoacids that are unusual for a human immunoglobulin at that position.These amino acids can be substituted with amino acids from theequivalent position of more typical human immunoglobulins.Alternatively, amino acids from equivalent positions in the mouseantibody can be introduced into the human framework regions when suchamino acids are typical of human immunoglobulin at the equivalentpositions.

In general, substitution of all or most of the amino acids fulfillingthe above criteria is desirable. Occasionally, however, there is someambiguity about whether a particular amino acid meets the abovecriteria, and alternative variant immunoglobulins are produced, one ofwhich has that particular substitution, the other of which does not.

The humanized antibodies of the invention that are derived from themouse 1D10 antibody usually contain a substitution of a human kappalight chain framework residue with a corresponding mu MAb 1D10 residuein at least 1, 2, 3 or 4 of the following positions: L48, L49, L69 andL70. The humanized antibodies also usually contain a substitution of ahuman heavy chain framework residue in at least 1, 2, 3, 4, 5, 6, 7, or8 of the following positions H27, H29, H30, H37, H67, H71, H78 and H83.In preferred embodiments when the human light chain acceptorimmunoglobulin is R3.5HG, the light chain also contains a substitutionat position 43. This position is substituted with the amino acid fromthe equivalent position of a human immunoglobulin having a more typicalamino acid residues or from a consensus sequence of such humanimmunoglobulins. Similarly, when the human heavy chain acceptorimmunoglobulin is IC4, the heavy chain also contains a substitution atposition 73.

The humanized antibodies of the invention that are derived from mouseM291 antibody contain no substitution of a human kappa light chainframework residue if the light chain acceptor is HF2-1/17. The humanizedantibodies also usually contain a substitution of a human heavy chainframework in at least 1, 2, 3, 4, 5 and 6 of the following positions:H30, H67, H68, H70, H72 and H74. In preferred embodiments, when theheavy chain acceptor immunoglobulin is 21/28, the light chain alsocontains a substitution at position 44. This position is substitutedwith the amino acid from the equivalent position of a humanimmunoglobulin having a more typical amino acid residue or from aconsensus sequence of such human immunoglobulin.

Usually the CDR regions in humanized antibodies are substantiallyidentical, and more usually, identical to the corresponding CDR regionsin the mouse antibody from which they were derived. Although not usuallydesirable, it is sometimes possible to make one or more conservativeamino acid substitutions of CDR residues without appreciably affectingthe binding affinity of the resulting humanized immunoglobulin.Occasionally, substitutions of CDR regions can enhance binding affinity.

Other than for the specific amino acid substitutions discussed above,the framework regions of humanized immunoglobulins are usuallysubstantially identical, and more usually, identical to the frameworkregions of the human antibodies from which they were derived. Of course,many of the amino acids in the framework region make little or no directcontribution to the specificity or affinity of an antibody. Thus, manyindividual conservative substitutions of framework residues can betolerated without appreciable change of the specificity or affinity ofthe resulting humanized immunoglobulin. However, in general, suchsubstitutions are undesirable.

(5) Production of Variable Regions

Having conceptually selected the CDR and framework components ofhumanized immunoglobulins, a variety of methods are available forproducing such immunoglobulins. Because of the degeneracy of the code, avariety of nucleic acid sequences will encode each immunoglobulin aminoacid sequence. The desired nucleic acid sequences can be produced by denovo solid-phase DNA synthesis or by PCR mutagenesis of an earlierprepared variant of the desired polynucleotide. All nucleic acidsencoding the antibodies described in this application are expresslyincluded in the invention.

(6) Selection of Constant Region

The variable segments of humanized antibodies produced as describedsupra are typically linked to at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Humanconstant region DNA sequences can be isolated in accordance withwell-known procedures from a variety of human cells, but preferablyimmortalized B-cells (see Kabat et al., supra, and WO87/02671).Ordinarily, the antibody will contain both light chain and heavy chainconstant regions. The heavy chain constant region usually includes CH1,hinge, CH2, CH3, and, sometimes, CH4 regions.

The humanized antibodies include antibodies having all types of constantregions, including IgM, IgG, IgD, IgA and IgE, and any isotype,including IgG1, IgG2, IgG3 and IgG4. When it is desired that thehumanized antibody exhibit cytotoxic activity, the constant domain isusually a complement-fixing constant domain and the class is typicallyIgG₁. When such cytotoxic activity is not desirable, the constant domainmay be of the IgG₂ class. The humanized antibody may comprise sequencesfrom more than one class or isotype.

(7) Expression Systems

Nucleic acids encoding humanized light and heavy chain variable regions,optionally linked to constant regions, are inserted into expressionvectors. The light and heavy chains can be cloned in the same ordifferent expression vectors. The DNA segments encoding immunoglobulinchains are operably linked to control sequences in the expressionvector(s) that ensure the expression of immunoglobulin polypeptides.Such control sequences include a signal sequence, a promoter, anenhancer, and a transcription termination sequence (see Queen et al.,Proc. Natl. Acad. Sci. USA 86, 10029 (1989); WO 90/07861; Co et al., J.Immunol. 148, 1149 (1992), which are incorporated herein by reference intheir entirety for all purposes).

C. Fragments of Humanized Antibodies

The humanized antibodies of the invention include fragments as well asintact antibodies. Typically, these fragments compete with the intactantibody from which they were derived for antigen binding. The fragmentstypically bind with an affinity of at least 10⁷ M⁻¹, and more typically10⁸ or 10⁹ M⁻¹ (i.e., within the same ranges as the intact antibody).Humanized antibody fragments include separate heavy chains, light chainsFab, Fab' F(ab')₂, and Fv. Fragments are produced by recombinant DNAtechniques, or by enzymic or chemical separation of intactimmunoglobulins.

Recombinant Bispecific Antibodies

The methods discussed above for forming bispecific antibodies fromantibodies produced by hybridoma cells can also be applied or adapted toproduction of bispecific antibodies from recombinantly expressedantibodies such as the humanized versions of 1D10 and M291. For example,bispecific antibodies can be produced by fusion of two cell linesrespectively expressing the component antibodies. Alternatively, thecomponent antibodies can be co-expressed in the same cell line.Bispecific antibodies can also be formed by chemical crosslinking ofcomponent recombinant antibodies.

Component recombinant antibodies can also be linked genetically. In oneapproach, a bispecific antibody is expressed as a single fusion proteincomprising the four different variable domains from the two componentantibodies separated by spacers. For example, such a protein mightcomprise from one terminus to the other, the VL region of the firstcomponent antibody, a spacer, the VH domain of the first componentantibody, a second spacer, the VH domain of the second componentantibody, a third spacer, and the VL domain of the second componentantibody. See, e.g., Segal et al., Biologic Therapy of Cancer Updates 2,1-12 (1992).

In a further approach, bispecific antibodies are formed by linkingcomponent antibodies to leucine zipper peptides. See generally copendingapplication 11823-003200 (Ser. No. 07/801,798, filed Nov. 29, 1991;Kostelny et al., J. Immunol. 148, 1547-1553 (1992) (incorporated byreference in their entirety for all purposes). Leucine zippers have thegeneral structural formula (Leucine-X₁ -X₂ -X₃ -X₄ -X₅ -X₆)_(n) (SEQ IDNO. 14), where X may be any of the conventional 20 amino acids(Proteins, Structures and Molecular Principles, (1984) Creighton (ed.),W. H. Freeman and Company, New York), but are most likely to be aminoacids with high α-helix forming potential, for example, alanine, valine,aspartic acid, glutamic acid, and lysine (Richardson and Richardson,Science 240, 1648 (1988)), and n may be 3 or greater, although typicallyn is 4 or 5. The leucine zipper occurs in a variety of eukaryoticDNA-binding proteins, such as GCN4, C/EBP, c-fos gene product (Fos),c-jun gene product (Jun), and c-myc gene product. In these proteins, theleucine zipper creates a dimerization interface wherein proteinscontaining leucine zippers may form stable homodimers and/orheterodimers.

The leucine zippers for use in the present invention preferably havepairwise affinity. Pairwise affinity is defined as the capacity for onespecies of leucine zipper, for example, the Fos leucine zipper, topredominantly form heterodimers with another species of leucine zipper,for example, the Jun leucine zipper, such that heterodimer formation ispreferred over homodimer formation when two species of leucine zipperare present in sufficient concentrations. See Schuemann et al., NucleicAcids Res. 19, 739 (1991). Thus, predominant formation of heterodimersleads to a dimer population that is typically 50 to 75 percent,preferentially 75 to 85 percent, and most preferably more than 85percent heterodimers. When amino-termini of the synthetic peptides eachinclude a cysteine residue to permit intermolecular disulfide bonding,heterodimer formation occurs to the substantial exclusion ofhomodimerization.

In the formation of bispecific antibodies, binding fragments of thecomponent antibodies are fused in-frame to first and second leucinezippers. Suitable binding fragments including Fv, Fab, Fab', or theheavy chain. The zippers can be linked to the heavy or light chain ofthe antibody binding fragment and are usually linked to the C-terminalend. If a constant region or a portion of a constant region is present,the leucine zipper is preferably linked to the constant region orportion thereof. For example, in a Fab'-leucine zipper fusion, thezipper is usually fused to the C-terminal end of the hinge. Theinclusion of leucine zippers fused to the respective component antibodyfragments promotes formation of heterodimeric fragments by annealing ofthe zippers. When the component antibodies include portions of constantregions (e.g., Fab' fragments), the annealing of zippers also serves tobring the constant regions into proximity, thereby promoting bonding ofconstant regions (e.g., in a F(ab')2 fragment). Typical human constantregions bond by the formation of two disulfide bonds between hingeregions of the respective chains. This bonding can be strengthened byengineering additional cysteine residue(s) into the respective hingeregions allowing formation of additional disulfide bonds.

Leucine zippers linked to antibody binding fragments can be produced invarious ways. For example, polynucleotide sequences encoding a fusionprotein comprising a leucine zipper can be expressed by a cellular hostor in vitro translation system. Alternatively, leucine zippers and/orantibody binding fragments can be produced separately, either bychemical peptide synthesis, by expression of polynucleotide sequencesencoding the desired polypeptides, or by cleavage from other proteinscontaining leucine zippers, antibodies, or macromolecular species, andsubsequent purification. Such purified polypeptides can be linked bypeptide bonds, with or without intervening spacer amino acid sequences,or by non-peptide covalent bonds, with or without intervening spacermolecules, the spacer molecules being either amino acids or othernon-amino acid chemical structures. Regardless of the method or type oflinkage, such linkage can be reversible. For example, a chemicallylabile bond, either peptidyl or otherwise, can be cleaved spontaneouslyor upon treatment with heat, electromagnetic radiation, proteases, orchemical agents. Two examples of such reversible linkage are: (1) alinkage comprising a Asn-Gly peptide bond which can be cleaved byhydroxylamine, and (2) a disulfide bond linkage which can be cleaved byreducing agents.

Component antibody fragment-leucine zippers fusion proteins can beannealed by co-expressing both fusion proteins in the same cell line.Alternatively, the fusion proteins can be expressed in separate celllines and mixed in vitro. If the component antibody fragments includeportions of a constant region (e.g., Fab' fragments), the leucinezippers can be cleaved after annealing has occurred. The componentantibodies remain linked in the bispecific antibody via the constantregions.

Therapeutic Methods

Pharmaceutical compositions comprising bispecific antibodies of thepresent invention are useful for parenteral administration, i.e.,subcutaneously, intramuscularly and particularly, intravenously. Thecompositions for parenteral administration commonly comprise a solutionof the antibody or a cocktail thereof dissolved in an acceptablecarrier, preferably an aqueous carrier. A variety of aqueous carrierscan be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine andthe like. These solutions are sterile and generally free of particulatematter. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, toxicity adjusting agents andthe like, for example sodium acetate, sodium chloride, potassiumchloride, calcium chloride, sodium lactate. The concentration of thebispecific antibodies in these formulations can vary widely, i.e., fromless than about 0.01%, usually at least about 0.1% to as much as 5% byweight and will be selected primarily based on fluid volumes, andviscosities in accordance with the particular mode of administrationselected.

A typical composition for intravenous infusion can be made up to contain250 ml of sterile Ringer's solution, and 10 mg of bispecific antibody.See Remington's Pharmaceutical Science (15th Ed., Mack PublishingCompany, Easton, Pa., 1980).

The compositions containing the present bispecific antibodies or acocktail thereof can be administered for prophylactic and/or therapeutictreatments. In therapeutic application, compositions are administered toa patient already affected by malignant B-cells (e.g., acutelymphoblastic leukemia, B-cell lymphoma, chronic lymphocytic leukemiaand multiple myeloma) in an amount sufficient to cure or at leastpartially arrest the condition and its complications. An amount adequateto accomplish this is defined as a "therapeutically effective dose."Amounts effective for this use will depend upon the severity of thecondition and the general state of the patient's own immune system, butgenerally range from about 0.01 to about 100 mg of bispecific antibodyper dose, with dosages of from 0.1 to 50 mg and 1 to 10 mg per patientbeing more commonly used. Single or multiple administrations on a daily,weekly or monthly schedule can be carried out with dose levels andpattern being selected by the treating physician.

In prophylactic applications, compositions containing the bispecificantibodies or a cocktail thereof are administered to a patient who is atrisk of developing the disease state to enhance the patient'sresistance. Such an amount is defined to be a "prophylacticallyeffective dose." In this use, the precise amounts again depend upon thepatient's state of health and general level of immunity, but generallyrange from 0.1 to 100 mg per dose, especially 1 to 10 mg per patient.

In some methods of treatment, bispecific antibodies are administeredwith a second agent (e.g., an interleukin) in an amount sufficient toactive effector cells thereby augmenting their cytotoxicity to malignantB-cells compared with the administration of bispecific antibody alone.Interleukin-2 at a dosage of about 500,000 U/kg is suitable. Combinationtherapy is particularly appropriate when the bispecific antibody beingadministered is a F(ab')2 fragment.

The monospecific 1D10antibody (particularly the humanized form) is alsosuitable for therapeutic administration to patients suffering from, orat risk of, B-cell malignancies. Optionally, the antibody is conjugatedto a radiolabel or toxin. The monospecific M291 antibody (particularlythe humanized form) can be used as an immunosuppressant in treatment ofdiseases and disorders of the immune system such as host vs. graftdisease, graft vs. host disease, autoimmune diseases, and inflammation.See, e.g,, Cosimi et al., N. Engl. J. Med. 305, 308 (1981); Russel etal., Annu. Rev. Med. 35, 63 (1984). The dosages and pharmaceuticalexcipients for administration of monospecific antibodies are similar tothose for the bispecific antibodies.

Diagnostic Methods

The M291 and 1D10 antibodies (both mouse and humanized forms) are alsouseful in diagnostic methods. The 1D10antibody (and other antibodiesbinding to the same or similar epitope) is useful for diagnosing thepresence of malignant B cells and monitoring the efficacy of treatmentsthereto. The antibody is also useful for research purposes to identifyand type cells of certain lineages and developmental origins. The M291antibody is useful for diagnostic purposes in immunologically monitoringof patients (see, e.g., Cosimi et al., supra) and for research purposesin classifying leukocyte subtypes, e.g., as part of an antibody panel.Methods of diagnosis can be performed in vitro using a cellular sample(e.g., blood sample, lymph node biopsy or tissue) from a patient or canbe performed by in vivo imaging.

EXAMPLE 1

The ability of 1DT3-D to induce the elimination of malignant B cells byT cells was evaluated in vitro. The assay used was a ⁵¹ chromium-releasecytotoxicity assay. Target malignant B cells (10⁷ cells in 1 ml) werelabeled during a 1 hour incubation with 100 μCi ⁵¹ Cr. T cells fromnormal donors were incubated in vitro with IL-2 or IL-2 and anti CD3antibody for 3-7 days before use as effector cells. T cells were addedto ⁵¹ Cr-labeled malignant B cells along with antibody. This mixture wasincubated for 4 hours, and cell free supernatant was removed andevaluated for the presence of released ⁵¹ Cr by gamma counting. Maximumrelease was determined by evaluating supernatant obtained from wellsthat had been treated with detergent (NP-40) that induces the lysis ofall cells. Background release was determined by evaluating ⁵¹ Cr levelsfrom samples that had target malignant B cells and T cells but noantibody. Specific release of ⁵¹ Cr indicated lysis of the ⁵¹Cr-containing target cells, and was calculated using the followingformula. ##EQU1##

FIG. 1 shows 1DT3-D induced the lysis of a large number of differentmalignant B cells including Raji (a cell line established from a patientwith Burkitt's lymphoma), HO-85 (a large cell lymphoma cell line), 697(a pre-B acute lymphoblastic leukemia cell line) and KH (freshlymphocytes obtained from a patient with chronic lymphocytic leukemia).The T-cell target cell ratio was 10:1 and the antibody concentration was5 μg/ml. Target lysis was not seen when monospecific antibody was used.

FIG. 2 shows 1DT3-D can induce significant lysis of raji cells at low Tcell: Raji cell ratios (less than 1:1) and at low antibodyconcentrations (less than 0.1 ug/ml. Similar results were seen withother target cell lines.

FIG. 3 shows 1DT3-D-induced T-cell-mediated lysis of fresh KH cells wasnoted after long incubation times.

The bispecific antibody of the present invention can also be producedsimply by taking the Fab or F(ab')₂ fragments of the 1D10 antibodyfusing these with portions of the OKT3 antibody to form a bispecificantibody of the present invention. Alternatively, bispecific antibodies,recognizing 1D10 and an antigen on natural killer cells or T cells, canbe produced, by synthetic or genetic engineering techniques.

A benefit of the claimed bispecific antibodies is their ability torecognize malignant B-cells and distinguish these from non-malignantB-cells. Thus, therapy using the bispecific antibodies of the presentinvention is significantly less damaging than therapy using, forexample, a non-specific antibody such as anti-CD19 antibody B4.

Further, as shown by the data described in the example, the antibody ofthe present invention induces significant lysis of malignant B-cells atrelatively low T cell ratios. FIG. 2 shows that malignant to T cellratios of less than 1:1 with relatively low antibody concentrations ofless than 0.1 micrograms per ml provides significant destruction of themalignant cells. This is particularly important since it reduces thedependency on the concentration of T cells available in the patient.Further, it also reduces the amount of antibody required, therebylimiting any potential side effects.

EXAMPLE 2 In Vivo Efficacy of 1DT3-D Bispecific Antibody

This example describes an in vivo trial of the bispecific antibody1DT3-D. Normal donor human peripheral blood lymphocytes were activatedin vitro in the presence of OKT3 (2 μg/ml), and recombinant IL-2 (300μg/ml). CB-17 scid/scid mice (Itoh et al., Cancer 72, 2686-2694 (1993))were injected subcutaneously with 5×10⁶ Raji cells mixed with 5×10⁶activated lymphocytes. 24-hr later, mice were injected with bispecificantibody, a monospecific antibody component of the bispecific antibodyor no antibody. Mice were examined daily for the development of tumorsof at least 0.5 cm at the site of tumor injection. Mice remainingtumor-free after 60 days were scored as negative and mice developingtumors within 60 days as positive. Control untreated mice alwaysdeveloped tumors within 21-28 days.

In a first experiment, 5 mice were treated with 10 μg/mouse ofbispecific antibody 24 hours after inoculation with the mixture ofmalignant cells and activated human T-cells. A control group of 5 micewas inoculated with vehicle only. The tumor occurrence (i.e.,development of a tumor of at least 0.5 cm within at least eight weeks)in the treated and control groups was as follows:

    ______________________________________                                        Group     No tumors     Tumors  Total                                         ______________________________________                                        Treatment 4             1       5                                             Control   0             5       5                                             ______________________________________                                    

Using the Fischer's one-sided exact test, bispecific antibody treatmentprolonged disease-free survival with a p value of 0.024.

A second experiment was designed to compare the anti-tumor effects ofbispecific antibody with monospecific anti-CD3 and monospecific 1D10 ata dose of 10 μg/mouse in mice inoculated with tumor and T-cells asoutlined above. Group 2 mice received monospecific 1D10 and monospecificOKT3, Group 3 received bispecific antibody and the control groupreceived vehicle only. Group 4 mice also received bispecific antibody ata concentration of 10 μg/mouse, but these mice had previously beeninoculated with unactivated T-cells as distinct from all other groupswhich received activated T-cells.

    ______________________________________                                        Group    No tumors      Tumors  Total                                         ______________________________________                                        Control  0              5       5                                             2        5              0       5                                             3        5              0       5                                             4        2              3       5                                             ______________________________________                                    

Fisher's exact test for general two-way tables (Agresti, CategoricalData Analysis (Wiley, N.Y., 1990), pp. 64-65) was used to test the nullhypothesis that the occurrence rates in the four groups are equal. Thereis a highly significant difference among the groups (p=0.001). Pairwiseexact tests comparing the control group to each of groups 2, 3, and 4were also carried out. The corresponding one-sides p-values are 0.004,0.004, and 0.222. Thus, groups 2 and 3 are both significantly differentfrom the control group. It was concluded that at a dose of 10 μg/mousetreatment with bispecific antibody or a combination of both componentmonospecific antibodies prolonged tumor-free survival.

In a third experiment, a dose-response study was performed to test theanti-tumor effects of varying dosages of bispecific antibody. Separategroups of mice were respectively treated with dosages of 0.4, 2 or 10μg/mouse bispecific antibody or vehicle.

    ______________________________________                                        Group    No tumors      Tumors  Total                                         ______________________________________                                        Control  0              5       5                                              0.4     1              4       5                                              2       5              0       5                                             10       4              1       5                                             ______________________________________                                    

The Cochran-Armitage trend test, (Agresti, Categorical Data Analysis(Wiley, N.Y., 1990), pp. 100-102, 118-119) was used to test the nullhypothesis that the occurrence rates in the four groups are equal,versus the alternative hypotheses of a linear trend. Usingequally-spaced scores, the p-value is 0.001; using the scores 0, 0.4, 2,and 10, the p-value is 0.0164. Both sets of scores indicate asignificant trend in the proportions. These results show that thebispecific antibody is effective to prolong survival time and that micereceiving larger doses (10 μg and 2 μg) have improved tumor-freesurvival.

A fourth experiment was designed to compare monospecific OKT3 and 1D10to bispecific antibody at a dose of 2 μg antibody/mouse.

    ______________________________________                                        Group    No tumors      Tumors  Total                                         ______________________________________                                        Control  0              5       5                                             2        0              5       5                                             3        1              4       5                                             4        4              1       5                                             ______________________________________                                    

Tumor-free survival of mice treated with monospecific OKT3 (Group 2) andmonospecific 1D10 (Group 3) was not significantly different fromcontrol, whereas mice treated with bispecific antibody (Group 4) hadprolonged survival using the Fisher's exact test for general two-waytables.

These data indicate that systemic administration of 1DT3-D kills and/orprevents the development of malignant B-cells in vivo and that a dose of2 μg/animal, bispecific antibody therapy is more effective thanmonospecific antibody therapy.

EXAMPLE 3 Generation of a Monoclonal Antibody Against the Human CD3Antigen

The 1DT3-D antibody described in Example 1 incorporated OKT3 as thebinding moiety having an affinity for effector cells. The presentexample describes the isolation of an alternative antibody, M291, foruse as the effector-cell binding component in a bispecific antibody.

Human peripheral blood mononuclear cells (PBMC) were activated with PHAand IL-2 to expand T cells. Activated T cells were used as immunogens inBalb/C mice. Hybridomas were generated from the spleens of these mice bystandard methods. These hybridomas were screened for antibodies thatcould stimulate PBMC to proliferate in vitro. Anti-CD3 antibodies withthe appropriate Fc cause T cells in PBMC to proliferate. One of thesehybridomas, M291, was isolated and found to secrete an antibody of theisotype IgG2a/kappa that could activate T cells to proliferate. Thepurified antibody M291 competes with another anti-human CD3 antibody,OKT3, (IgG2a/kappa) for binding to human T cells, showing that theepitopes recognized by the respective antibodies are closely spaced.M291 is thus an antibody having the specificity against the human CD3complex.

EXAMPLE 4 Humanization of 1D10 and M291 Antibodies

This example describes the separate humanization procedures for the 1D10and M291 antibodies.

(1) Cloning of 1D10 and M291 V region cDNAs

Heavy and light V domain cDNAs for 1D10and M291 were cloned using ananchored PCR method (see Loh et al., Science 243, 217 (1989)). cDNAswere first synthesized by reverse transcriptase after priming polyA+RNAs from the hybridoma cells with oligo dT. A tail of dGs was added tothe 3' terminus of the cDNA by terminal deoxynucleotidyl transferase.The V domains were then amplified by PCR with 3' primers that hybridizedto the C regions and 5' primers that hybridized to the G-tails. Severalindependent H and L chain clones were sequenced to ensure no sequencemistakes were introduced by PCR. For 1D10, the V domains were expressedas an antibody of the mouse isotype IgG2a/kappa by transfecting thegenes in suitable vectors into the myeloma cell line SP2/0 to confirmthey coded for the binding site of 1D10. The expression vectors used inthe transfection are similar to the plasmids pVk.G and pVg.D describedby Co et al. (see Co et al., J. Immunol. 148, 1149 (1992)), except thatgenes for the constant regions were derived from mouse sequences.Antibody isolated from the transfected cells was found by flow cytometryto bind to Raji cells in a pattern indistinguishable from that of theparent mouse IgG1/kappa 1D10 antibody. The V domains of M291 were clonedsimilarly and they were expressed as mouse F(ab'-zipper)₂ (see Kostelnyet al., J. Immunol. 148, 1547 (1992)). Flow cytometry assay indicatedM291-Fos F(ab'zipper)₂ binds to human T cells with similar or identicalaffinity as the parent antibody. This observation confirmed that thecorrect V domains of M291 were cloned.

(2) Modelling and design of humanized sequences

The sequences of human V domains most similar to murine 1D10 and M291were selected to serve as the framework of the humanized antibody. For1D10, the best human V_(k) sequence was R3.5H5G of human subgroup I withonly sixteen differences from 1D10in framework regions. Manheimer-Loryet al., J. Exp. Med. 174, 1639-1652 (1991). The best V_(H) sequence wasIC4 of Kabat's subgroup II or subgroup IV (see Id.), with twenty-sixdifferences. For M291, the best human V_(k) sequence is HF2-1/17 ofhuman subgroup I with twenty-six amino acid differences from M291 inframework regions (Athison et al., J. Clin. Invest. 75, 1138 (1985);Lampman Blood 74, 262 (1989)); the best human V_(H) sequence is 21/28 ofhuman subgroup I with twenty amino acid differences. Dersimonian et al.,J. Immunol. 139, 2496-2501 (1987). With the help of the 3-dimensionalmodel, an additional number of framework positions that differed betweenthe murine antibodies and the chosen human sequences were identified.The location of those amino acid residues in 3-dimensional spacerelative to the hypervariable regions, or CDRs, indicated they werelikely to influence CDR conformation, and thus binding affinity. Murinesequences were used in these positions. A number of positions wereidentified in the human sequences that differed from the consensus oftheir respective subgroups. These amino acids were changed to correspondto consensus sequences. V_(H) and V_(L) sequence comparisons between themurine and humanized 1D10, and between the murine and humanized M291,are shown in FIG. 4 and FIG. 5, respectively.

(3) Synthesis and expression of humanized 1D10 antibody

DNA segments encoding the humanized 1D10 L and H chain V regions wereconstructed by total gene synthesis from overlapping oligonucleotides.These mini exons included signal sequences, J segments and splice donorsequences and were surrounded by XbaI sites. The DNA segments wereincorporated in an expression vector using the scheme outlined in FIG.6.

The humanized V domains were cloned into the XbaI sites of thecorresponding heavy and light chain expression plasmids pV_(g) 1.D.Ttand pVk.rG.dE. The resulting plasmids are called pHu1D10.Vg1.D.Tt andpHu1D10.V_(k).rG.dE. The heavy chain expression vector, pVg1.D.Tt, whichcontains the mutant dihydrofolate reductase gene (mdhfr) as theselectable marker (see Simonsen & Levinson, Proc. Natl. Acad. Sci. USA,80, 2495, (1983)), the human cytomegalovirus (hCMV) major immediateearly promoter and enhancer for transcription initiation (see Boshart etal., Cell 41, 521 (1985)), and the human IgG1 constant regions wasconstructed from the respective fragments by standard methods. Itdiffers from the vector pV_(g) 1.D described by Co et al, J. Immunol.148, 1149 (1992) by having a transcription termination site 3' to the γ1 gene poly(A) site. The transcription termination site (Tt) was derivedfrom the sequence located downstream from the human complement gene C2(+37 to +162 bp from the C2 poly(A) site) (see Ashfield et al., EMBO J.10, 4197 (1991)) and was synthesized entirely by using overlappingoligonucleotides.

For light chain expression, a vector was constructed from the hCMVpromoter and enhancer, the human C_(K) gene including part of thepreceding intron, and the xanthine-guanine phosphoribosyltransferase(gpt) gene (see Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78, 2072(1981)) for selection. The vector, pV_(k).rG.dE, is similar to pV_(k)described by Co et al. (see Co et al., J. Immunol. 148, 1149 (1992))except for the orientation of the gpt gene. In addition, one of the tworepeated sequences in the enhancer region of the SV40 promoter used totranscribe the gpt gene was deleted by SphI digestion.

For coexpression of heavy and light chains in one plasmid, an EcoRIfragment containing the hCMV promoter, the V_(H) exon, the C_(H) 1,C_(H) 2 and C_(H) 3 exons, the polyA signal, and the transcriptiontermination signal was taken from the heavy chain expression vector andcloned into the unique EcoRI site of the corresponding light chainexpression plasmid. Due to the presence of the transcription terminationsignal situated between them, the two genes are transcribedindependently by the hCMV promoter. After transcription the humanizedV_(H) exon is spliced to the human γ 1 C_(H) 1, hinge, C_(H) 2 and C_(H)3 exons, and then polyadenylated. Similarly the V_(L) exon is spliced tothe human C_(K) exon. The predicted amino acid sequences of the maturelight and heavy chains of humanized 1D10 are shown in FIGS. 4C and 4E,respectively.

Plasmid pHu1D10.IgG1.rG.dE, was used for transfection into mouse myelomacell line TSO by electroporation. TSO cells are derivative of mousemyeloma NSO cells (ECACC 85110503) selected for their ability to grow inserum-free media according to the procedure of Sato et al., J. Exp. Med.165, 1761 (1987). The cells from each transfection were selected for gptexpression. Because the SV40 promoter/enhancer for the gpt gene has beencrippled, only few transfectants can express gpt high enough to survivethe selection (see Jasin & Berg, Genes Dev. 2, 1353 (1988)).Transfection efficiency is about 0.5-1.0×10⁻⁶ ; compared to theefficiency of 10-50×10⁻⁶ from transfection using near identical plasmidcontaining the wild type SV40 promoter for gpt. When screened forproduction of humanized antibodies by standard ELISA, the averagesurviving cells also gave higher levels of antibody compared to thosetransfected with plasmid containing the wild type SV40 promoter. Thebest antibody producer was then subcloned for the production of thehumanized 1D10. The antibody, Hu1D10, was purified from the serum-freespent medium by Protein A affinity chromatography.

(4) Properties of Hu1D10

Murine 1D10-IgG2a and humanized 1D10 had identical spectrums ofreactivity with 1D10 positive and 1D10 negative cell lines. The affinityof murine 1D10-IgGa and humanized 1D10 for cells bearing the targetantigen was evaluated using a displacement assay (see Woodle et al., J.Immunol. 148 2756 (1992)). In this assay, the ability of preboundhumanized 1D10 or murine 1D10-IgG2a to inhibit the binding ofFITC-labeled murine 1D10-IgG2a was quantitated by FACS analysis.Humanized 1D10 competitively inhibited the binding of murine 1D10-IgG2ato a degree similar to that seen with the parent antibody (FIG. 7A).These data indicated that the humanized antibody binds with similaraffinity as the murine antibody. Scatchard analysis was used to betterestimate the apparent affinity of humanized 1D10. Humanized 1D10-IgG1was found to have an apparent K_(a) of 2.3×10⁸ M⁻¹, and there are about5×10⁵ sites per cell in the Raji cell line (FIG. 7B). In addition,humanized 1D10 has the ability to direct ADCC and complement mediatedlysis, two effector functions that are not present in the originalmurine 1D10 (FIGS. 8A and 8B).

(5) Synthesis and expression of humanized M291 and 1D10(ab'-zipper)₂

Leucine zipper genes, Jun and Fos, were synthesized as described byKostelny et al., J. Immunol. 148, 1547 (1992). The resulting PCRproducts were 179 bp PstI-SalI fragments, encompassing the entire hingezipper gene fusion. The PstI site is the natural restriction sitelocated at the beginning of the hinge exon, but the SalI site was addedto the end of the zipper sequences during PCR. The hinge/zipper exon wasinserted with a 162 bp SalI-BamHI fragment containing the 3' noncodingsequence of the mouse IgG2a gene into the heavy chain expression vectorpVg1.D.Tt, replacing the hinge, C_(H) 2 and C_(H) 3 exons in theplasmid. Coexpression of the truncated heavy chain (Fd) gene with lightchain gene in one plasmid is essentially the same as described above forpHu1D10.IgG1.rG.dE (FIG. 6). The expression plasmids are calledpHu1D10-Jun.rG.dE and pHuM291-Fos.rG.dE (FIG. 9). The differencesbetween these plasmids and those used to express the whole antibody are:(1) the human γ1 C_(H) 1 exon is now spliced to the hinge/zipper fusionexon instead of the hinge, C_(H) 2 and C_(H) 3 exons, and (2) thetranscript is polyadenylated by a heterologous signal. The leucinezipper Jun is used for the Fd of Hu1D10, and Fos for Fd of HuM291. Whencombined with the corresponding light chain, the Fd-zipper would formF(ab'-zipper)₂. The humanized F(ab'-zipper)₂ fragments for 1D10 and M291are called Hu1D10-Jun and HuM291-Fos, respectively. The predicted aminoacid sequences of the heavy chain Fd-zipper in Hu1D10-Jun and HuM291-Fosare shown in FIGS. 4D and 5D, respectively. In both cases there weremodifications of the human IgG1 hinge at the region of hinge/zipperfusion (FIG. 10). An insertion of two amino acid residues Lys-Cysderived from the mouse IgG2a hinge was introduced to the hinge exon toprovide an additional inter-heavy chain disulfide bond. The insertion ofthese two residues in the human IgG1 hinge renders its COOH-terminalhalf homologous to that of the mouse IgG2a hinge. The modified hingewould have three inter-heavy chain disulfide bonds compared to two inthe wild type human IgG1. In addition an Ala residue (first residue ofthe C_(H) 2 domain) and two Gly residues were introduced at the fusionjunction to make the joints more flexible. The expression plasmids,pHu1D10-Jun.rG.dE and pHuM291-Fos.rG.dE, were separately transfectedinto mouse myeloma cell line TSO by electroporation. Transfectants werescreened for the presence and the quantity of secreted F(ab'-zipper)₂fragments by ELISA. F(ab'-zipper)₂ fragments were purified using ProteinG affinity chromatography.

(5) Properties of HuM291-Fos

The relative affinity of murine M291 and HuM291-Fos F(ab'-zipper)₂ for Tcells was evaluated using the displacement assay described above.HuM291-Fos blocks the binding of FITC-labeled murine M291 IgG2a as wellas the unlabeled M291 (FIG. 11A). The affinity of HuM291 for CD3 isestimated to be within 2-3 fold of M291's. Scatchard analysis indicatedthe apparent affinity of HuM291-Fos was K_(a) .sup.˜ 1.1×10⁹ M⁻¹, andthere are about 6.6×10⁴ sites per cell in activated human T cells (FIG.11B).

(6) Formation of the bispecific Hu1D10-Jun×HuM291-Fos F(ab'-zipper)₂ invitro

Hu1D10-Jun and HuM291-Fos were mixed in equal molar at concentrationsbetween 0.5 to 3.0 mg/ml and reduced with 10 mM DTT in PBS at 37° C. for1 hour to form Fab'-zippers. They were passed through Sepharose G-50column in PBS to remove DTT. The desalted protein was incubated at 4° C.for 48 hours to allow formation of heterodimeric bispecific Hu1D10-Jun×HuM291-Fos. The bispecific molecules were further purified byhydrophobic interaction chromatography (HIC) on a Phenyl Sepharosecolumn.

EXAMPLE 5 T Cell-mediated Cytotoxicity by Humanized BispecificAntibodies

The ability of Hu1D10-Jun×HuM291-Fos to direct T cell-mediated lysis wastested in a chromium-release assay. Human T cells derived from PBMCafter OKT3 and IL-2 treatment were used as effector cells. Dawo, whichis a cell line developed from a patient with large B cell lymphoma, wasused as target cells. FIG. 12 shows that the bispecificHu1D10-Jun×HuM291-Fos, as well as the mouse bispecific IgG 1DT3-Ddirected T cells to lyse target cells. The two bispecific moleculesseemed to have similar activities at low antibody concentrations. Thetwo parent antibodies, HuM291-Fos and Hu1D10-Jun, were not effective inthis assay, either singly or in combination.

At high concentrations (10 μg/ml, 1DT3-D had higher activity thanHu1D10-Jun×HuM291-Fos in mediating target cell lysis. This was becauseof low affinity Fc receptors on the surface of the target cells. At highantibody concentration, bispecific IgG's Fc could bind to thesereceptors and direct T cell to lyse target cells independent of thetarget antigen, a mechanism known as reverse lysis (see Weiner et al.,J. Immunol. 152, 2385 (1994)). Because Hu1D10-Jun×HuM291-Fos is anF(ab')₂ -like molecule without an Fc, it cannot initiate lysis bybinding to an Fc receptor. In some therapeutic application, the propertyof the humanized antibody is advantageous in increasing selectivetoxicity of the antibody.

All publications and patent applications cited above are hereinincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent application werespecifically and individually indicated to be so incorporated byreference. Although the present invention has been described in somedetail by way of illustration and example for purposes of clarity andunderstanding, it will be apparent that certain changes andmodifications may be practiced within the scope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 14                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 107 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Le - #u Ser Ala Ser Val Gly         #                15                                                           - Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gl - #u Asn Ile Tyr Ser Tyr         #            30                                                               - Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Al - #a Pro Lys Leu Leu Val         #        45                                                                   - Ser Asn Ala Lys Thr Leu Ala Glu Gly Val Pr - #o Ser Arg Phe Ser Gly         #    60                                                                       - Ser Gly Ser Gly Lys Gln Phe Thr Leu Thr Il - #e Ser Ser Leu Gln Pro         #80                                                                           - Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Hi - #s Tyr Gly Asn Ser Tyr         #                95                                                           - Pro Phe Gly Gln Gly Thr Lys Leu Glu Ile Ly - #s                             #           105                                                               - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 107 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Le - #u Ser Ala Ser Val Gly         #                15                                                           - Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gl - #u Asn Ile Tyr Ser Tyr         #            30                                                               - Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Se - #r Pro Gln Leu Leu Val         #        45                                                                   - Ser Asn Ala Lys Thr Leu Ala Glu Gly Val Th - #r Ser Arg Phe Ser Gly         #    60                                                                       - Ser Gly Ser Gly Lys Gln Phe Ser Leu Lys Il - #e Asn Ser Leu Gln Pro         #80                                                                           - Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Hi - #s Tyr Gly Asn Ser Tyr         #                95                                                           - Pro Phe Gly Gly Gly Thr Lys Leu Glu Ile Ly - #s                             #           105                                                               - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 116 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Le - #u Val Lys Pro Ser Glu         #                15                                                           - Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ph - #e Ser Leu Thr Asn Tyr         #            30                                                               - Gly Val His Trp Val Arg Gln Ser Pro Gly Ly - #s Gly Leu Glu Trp Ile         #        45                                                                   - Gly Val Lys Trp Ser Gly Gly Ser Thr Glu Ty - #r Asn Ala Ala Phe Ile         #    60                                                                       - Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ly - #s Asn Gln Val Ser Leu         #80                                                                           - Lys Leu Asn Ser Leu Thr Ala Ala Asp Thr Al - #a Val Tyr Tyr Cys Ala         #                95                                                           - Arg Asn Asp Arg Tyr Ala Met Asp Tyr Trp Gl - #y Gln Gly Thr Leu Val         #           110                                                               - Thr Val Ser Ser                                                                     115                                                                   - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 116 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Le - #u Val Gln Pro Ser Gln         #                15                                                           - Ser Leu Ser Ile Thr Cys Thr Gly Ser Gly Ph - #e Ser Leu Thr Asn Tyr         #            30                                                               - Gly Val His Trp Val Arg Gln Ser Pro Gly Ly - #s Gly Leu Glu Trp Ile         #        45                                                                   - Gly Val Lys Trp Ser Gly Gly Ser Thr Glu Ty - #r Asn Ala Ala Phe Ile         #    60                                                                       - Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Ly - #s Ser Gln Val Phe Phe         #80                                                                           - Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Al - #a Met Tyr Tyr Cys Ala         #                95                                                           - Arg Asn Asp Arg Tyr Ala Met Asp Tyr Trp Gl - #y Gln Gly Thr Ser Val         #           110                                                               - Thr Val Ser Ser                                                                     115                                                                   - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 214 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Le - #u Ser Ala Ser Val Gly         #                15                                                           - Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gl - #u Asn Ile Tyr Ser Tyr         #            30                                                               - Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Al - #a Pro Lys Leu Leu Val         #        45                                                                   - Ser Asn Ala Lys Thr Leu Ala Glu Gly Val Pr - #o Ser Arg Phe Ser Gly         #    60                                                                       - Ser Gly Ser Gly Lys Gln Phe Thr Leu Thr Il - #e Ser Ser Leu Gln Pro         #80                                                                           - Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Hi - #s Tyr Gly Asn Ser Tyr         #                95                                                           - Pro Phe Gly Gln Gly Thr Lys Leu Glu Ile Ly - #s Arg Thr Val Ala Ala         #           110                                                               - Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Gl - #u Gln Leu Lys Ser Gly         #       125                                                                   - Thr Ala Ser Val Val Cys Leu Leu Asn Asn Ph - #e Tyr Pro Arg Glu Ala         #   140                                                                       - Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gl - #n Ser Gly Asn Ser Gln         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Se - #r Thr Tyr Ser Leu Ser         #               175                                                           - Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Gl - #u Lys His Lys Val Tyr         #           190                                                               - Ala Cys Glu Val Thr His Gln Gly Leu Ser Se - #r Pro Val Thr Lys Ser         #       205                                                                   - Phe Asn Arg Gly Glu Cys                                                         210                                                                       - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 273 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Le - #u Val Lys Pro Ser Glu         #                15                                                           - Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ph - #e Ser Leu Thr Asn Tyr         #            30                                                               - Gly Val His Trp Val Arg Gln Ser Pro Gly Ly - #s Gly Leu Glu Trp Ile         #        45                                                                   - Gly Val Lys Trp Ser Gly Gly Ser Thr Glu Ty - #r Asn Ala Ala Phe Ile         #    60                                                                       - Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ly - #s Asn Gln Val Ser Leu         #80                                                                           - Lys Leu Asn Ser Leu Thr Ala Ala Asp Thr Al - #a Val Tyr Tyr Cys Ala         #                95                                                           - Arg Asn Asp Arg Tyr Ala Met Asp Tyr Trp Gl - #y Gln Gly Thr Leu Val         #           110                                                               - Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Se - #r Val Phe Pro Leu Ala         #       125                                                                   - Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Al - #a Ala Leu Gly Cys Leu         #   140                                                                       - Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Va - #l Ser Trp Asn Ser Gly         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ala Leu Thr Ser Gly Val His Thr Phe Pro Al - #a Val Leu Gln Ser Ser         #               175                                                           - Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Va - #l Pro Ser Ser Ser Leu         #           190                                                               - Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn Hi - #s Lys Pro Ser Asn Thr         #       205                                                                   - Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cy - #s Asp Lys Thr His Thr         #   220                                                                       - Cys Pro Pro Cys Lys Cys Pro Ala Gly Gly Ar - #g Ile Ala Arg Leu Glu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Glu Lys Val Lys Thr Leu Lys Ala Gln Asn Se - #r Glu Leu Ala Ser Thr         #               255                                                           - Ala Asn Met Leu Arg Glu Gln Val Ala Gln Le - #u Lys Gln Lys Val Met         #           270                                                               - Asn                                                                         - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 446 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Le - #u Val Lys Pro Ser Glu         #                15                                                           - Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ph - #e Ser Leu Thr Asn Tyr         #            30                                                               - Gly Val His Trp Val Arg Gln Ser Pro Gly Ly - #s Gly Leu Glu Trp Ile         #        45                                                                   - Gly Val Lys Trp Ser Gly Gly Ser Thr Glu Ty - #r Asn Ala Ala Phe Ile         #    60                                                                       - Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ly - #s Asn Gln Val Ser Leu         #80                                                                           - Lys Leu Asn Ser Leu Thr Ala Ala Asp Thr Al - #a Val Tyr Tyr Cys Ala         #                95                                                           - Arg Asn Asp Arg Tyr Ala Met Asp Tyr Trp Gl - #y Gln Gly Thr Leu Val         #           110                                                               - Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Se - #r Val Phe Pro Leu Ala         #       125                                                                   - Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Al - #a Ala Leu Gly Cys Leu         #   140                                                                       - Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Va - #l Ser Trp Asn Ser Gly         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ala Leu Thr Ser Gly Val His Thr Phe Pro Al - #a Val Leu Gln Ser Ser         #               175                                                           - Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Va - #l Pro Ser Ser Ser Leu         #           190                                                               - Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn Hi - #s Lys Pro Ser Asn Thr         #       205                                                                   - Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cy - #s Asp Lys Thr His Thr         #   220                                                                       - Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gl - #y Gly Pro Ser Val Phe         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Me - #t Ile Ser Arg Thr Pro         #               255                                                           - Glu Val Thr Cys Val Val Val Asp Val Ser Hi - #s Glu Asp Pro Glu Val         #           270                                                               - Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Va - #l His Asn Ala Lys Thr         #       285                                                                   - Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Ty - #r Arg Val Val Ser Val         #   300                                                                       - Leu Thr Val Leu His Gln Asp Trp Leu Asn Gl - #y Lys Glu Tyr Lys Cys         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Il - #e Glu Lys Thr Ile Ser         #               335                                                           - Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Va - #l Tyr Thr Leu Pro Pro         #           350                                                               - Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Se - #r Leu Thr Cys Leu Val         #       365                                                                   - Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Gl - #u Trp Glu Ser Asn Gly         #   380                                                                       - Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pr - #o Val Leu Asp Ser Asp         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Va - #l Asp Lys Ser Arg Trp         #               415                                                           - Gln Gln Gly Asn Val Phe Ser Cys Ser Val Me - #t His Glu Ala Leu His         #           430                                                               - Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Se - #r Pro Gly Lys                 #       445                                                                   - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 106 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Le - #u Ser Ala Ser Val Gly         #                15                                                           - Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Se - #r Ser Val Ser Tyr Met         #            30                                                               - Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pr - #o Lys Arg Leu Ile Tyr         #        45                                                                   - Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Se - #r Arg Phe Ser Gly Ser         #    60                                                                       - Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Se - #r Ser Leu Gln Pro Glu         #80                                                                           - Asp Phe Asp Thr Tyr Tyr Cys Gln Gln Trp Se - #r Ser Asn Pro Pro Thr         #                95                                                           - Phe Gly Gly Gly Thr Lys Val Glu Ile Lys                                     #           105                                                               - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 106 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Me - #t Ser Ala Ser Pro Gly         #                15                                                           - Glu Lys Val Thr Met Thr Cys Ser Ala Ser Se - #r Ser Val Ser Tyr Met         #            30                                                               - Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pr - #o Lys Arg Trp Thr Tyr         #        45                                                                   - Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Al - #a Arg Phe Ser Gly Ser         #    60                                                                       - Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Se - #r Ser Met Glu Ala Glu         #80                                                                           - Asp Ala Asp Thr Tyr Tyr Cys Gln Gln Trp Se - #r Ser Asn Pro Pro Thr         #                95                                                           - Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys                                     #           105                                                               - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 120 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Gln Val Gln Leu Val Gln Ser Gly Ala Glu Va - #l Lys Lys Pro Gly Ala         #                15                                                           - Ser Val Lys Val Ser Cys Lys Ala Ser Gly Ty - #r Thr Phe Ile Ser Tyr         #            30                                                               - Thr Met His Trp Val Arg Gln Ala Pro Gly Gl - #n Gly Leu Glu Trp Met         #        45                                                                   - Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Thr Hi - #s Tyr Asn Gln Lys Leu         #    60                                                                       - Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Se - #r Ala Ser Thr Ala Tyr         #80                                                                           - Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Th - #r Ala Val Tyr Tyr Cys         #                95                                                           - Ala Arg Ser Ala Tyr Tyr Asp Tyr Asp Gly Ph - #e Ala Tyr Trp Gly Gln         #           110                                                               - Gly Thr Leu Val Thr Val Ser Ser                                             #       120                                                                   - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 120 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Le - #u Ala Arg Pro Gly Ala         #                15                                                           - Ser Val Lys Met Ser Cys Lys Ala Ser Gly Ty - #r Thr Phe Ile Ser Tyr         #            30                                                               - Thr Met His Trp Val Lys Gln Arg Pro Gly Gl - #n Gly Leu Glu Trp Ile         #        45                                                                   - Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Thr Hi - #s Tyr Asn Gln Lys Leu         #    60                                                                       - Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Se - #r Ser Ser Ser Ala Tyr         #80                                                                           - Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Se - #r Ala Val Tyr Tyr Cys         #                95                                                           - Ala Arg Ser Ala Tyr Tyr Asp Tyr Asp Gly Ph - #e Ala Tyr Trp Gly Gln         #           110                                                               - Gly Thr Leu Val Thr Val Ser Ala                                             #       120                                                                   - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 213 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Le - #u Ser Ala Ser Val Gly         #                15                                                           - Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Se - #r Ser Val Ser Tyr Met         #            30                                                               - Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pr - #o Lys Arg Leu Ile Tyr         #        45                                                                   - Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Se - #r Arg Phe Ser Gly Ser         #    60                                                                       - Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Se - #r Ser Leu Gln Pro Glu         #80                                                                           - Asp Phe Asp Thr Tyr Tyr Cys Gln Gln Trp Se - #r Ser Asn Pro Pro Thr         #                95                                                           - Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ar - #g Thr Val Ala Ala Pro         #           110                                                               - Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gl - #n Leu Lys Ser Gly Thr         #       125                                                                   - Ala Ser Val Val Cys Leu Leu Asn Asn Phe Ty - #r Pro Arg Glu Ala Lys         #   140                                                                       - Val Gln Trp Lys Val Asp Asn Ala Leu Gln Se - #r Gly Asn Ser Gln Glu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Th - #r Tyr Ser Leu Ser Ser         #               175                                                           - Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Ly - #s His Lys Val Tyr Ala         #           190                                                               - Cys Glu Val Thr His Gln Gly Leu Ser Ser Pr - #o Val Thr Lys Ser Phe         #       205                                                                   - Asn Arg Gly Glu Cys                                                             210                                                                       - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 279 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - Gln Val Gln Leu Val Gln Ser Gly Ala Glu Va - #l Lys Lys Pro Gly Ala         #                15                                                           - Ser Val Lys Val Ser Cys Lys Ala Ser Gly Ty - #r Thr Phe Ile Ser Tyr         #            30                                                               - Thr Met His Trp Val Arg Gln Ala Pro Gly Gl - #n Gly Leu Glu Trp Met         #        45                                                                   - Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Thr Hi - #s Tyr Asn Gln Lys Leu         #    60                                                                       - Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Se - #r Ala Ser Thr Ala Tyr         #80                                                                           - Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Th - #r Ala Val Tyr Tyr Cys         #                95                                                           - Ala Arg Ser Ala Tyr Tyr Asp Tyr Asp Gly Ph - #e Ala Tyr Trp Gly Gln         #           110                                                               - Gly Thr Leu Val Thr Val Ser Ser Ala Ser Th - #r Lys Gly Pro Ser Val         #       125                                                                   - Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Se - #r Gly Gly Thr Ala Ala         #   140                                                                       - Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Gl - #u Pro Val Thr Val Ser         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Trp Asn Ser Gly Ala Leu Thr Ser Gly Val Hi - #s Thr Phe Pro Ala Val         #               175                                                           - Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Se - #r Val Val Thr Val Pro         #           190                                                               - Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cy - #s Asn Val Asn His Lys         #       205                                                                   - Pro Ser Asn Thr Lys Val Asp Lys Lys Val Gl - #u Pro Lys Ser Cys Asp         #   220                                                                       - Lys Thr His Thr Cys Pro Pro Cys Lys Cys Pr - #o Ala Gly Gly Leu Thr         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Asp Thr Leu Gln Ala Glu Thr Asp Gln Leu Gl - #u Asp Lys Lys Ser Ala         #               255                                                           - Leu Gln Thr Glu Ile Ala Asn Leu Leu Lys Gl - #y Lys Glu Lys Leu Glu         #           270                                                               - Phe Ile Leu Ala Ala Thr Ser                                                         275                                                                   - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - Leu Xaa Xaa Xaa Xaa Xaa Xaa                                                 1               5                                                             __________________________________________________________________________

What is claimed is:
 1. A bispecific antibody that binds to:(a) a firstantigen on the surface of effector cells selected from the groupconsisting of T-cells and natural killer cells, and (b) a second antigenon a 28/32 kDa heterodimeric protein on the surface of malignant Bcells, which second antigen specifically binds to an antibody designated1D10, having a light chain variable domain designated SEQ. ID. No. 2 anda heavy chain variable domain designated SEQ. ID. No. 4, wherein thebinding of the bispecific antibody to the first and second antigensresults in killing of the malignant B cells.
 2. The bispecific antibodyof claim 1, wherein the effector cells are T cells.
 3. The bispecificantibody of claim 2, wherein the bispecific antibody binds to a CD3antigen on the T cells.
 4. The bispecific antibody of claim 3, whereinthe bispecific antibody is produced by the cell line ATCC HB 1099.3. 5.A cell line producing the bispecific antibody of claim
 1. 6. The cellline of claim 5 that is a hybrid-hybridoma formed from two hybridomas,afirst hybridoma producing an antibody that binds to the first antigen,and a second hybridoma producing an antibody that binds to the secondantigen.
 7. The cell line of claim 6, wherein the effector cells areT-cells and the first hybridoma produces an antibody that binds to a CD3antigen on the T-cells.
 8. A cell line designated ATCC HB
 10993. 9. Anantibody designated 1D10, having a light chain variable domaindesignated SEQ. ID. No. 2 and a heavy chain variable domain designatedSEQ. ID. No.
 4. 10. A humanized version of the antibody of claim
 9. 11.A humanized antibody according to claim 10, the antibody comprising ahumanized heavy chain and a humanized light chain:(1) the humanizedlight chain comprising three complementarity determining regions, CDR1,CDR2 and CDR3, of the 1D10 immunoglobulin light chain, and a variableregion framework from a human kappa light chain variable regionframework sequence provided that at least one position selected from afirst group consisting of L48, L49, L69, and L70 is occupied by the sameamino acid present in the equivalent position of the 1D10 immunoglobulinlight chain variable region framework; and (2) the humanized heavy chaincomprising three complementarity determining regions, CDR1, CDR2 andCDR3, of 1D10 immunoglobulin heavy chain, and a variable regionframework from a human heavy chain variable region framework sequenceprovided that at least one position selected from a second groupconsisting of H27, H29, H30, H37, H67, H71, H78 and H83 is occupied bythe same amino acid present in the equivalent position of the mouse 1D10immunoglobulin heavy chain variable region framework; wherein thehumanized antibody specifically binds to a 28/32 kDa heterodimericprotein on the surface of malignant B cells with a binding affinityhaving a lower limit of about 10⁷ M⁻¹ and an upper limit of aboutfive-times the binding affinity of the 1D10 immunoglobulin.
 12. Thehumanized antibody of claim 11, whereinthe humanized light chainvariable region framework is from the light chain variable regionframework of the R3.5H5G antibody provided that at least one positionfrom the first group is occupied by the same amino acid present in theequivalent Position of the 1D10 immunoglobulin light chain variableregion framework and provided that position L43 is occupied by the aminoacid present in the equivalent position of a human kappa subgroup Iconsensus sequence; the humanized heavy chain variable region frameworkis from the heavy chain region framework of the IC4 antibody provided atleast one position selected from the second group is occupied by thesame amino acid present in the equivalent position of the mouse1D10immunoglobulin heavy chain variable region framework, and providedthat position H73 is occupied by the same amino acid present in theequivalent position of a human immunoglobulin subgroup II or IVconsensus sequence.
 13. The humanized antibody of claim 12, whereinthehumanized light chain comprises the amino acid sequence of FIG. 4A(upper) (SEQ ID NO. 1) and the humanized heavy chain comprises the aminoacid sequence of FIG. 4B (upper) (SEQ ID NO. 3).
 14. The humanizedantibody of claim 13, wherein the humanized light chain furthercomprises a human kappa constant region, the humanized heavy chainfurther comprises a human γ1 constant region, and the humanized antibodyeffects ADCC and complement-mediated lysis of malignant B-cells whenbound to a 28/32 kDa heterodimeric protein on the surface of the cells.15. A humanized antibody, the antibody comprising a humanized heavychain and a humanized light chain:(1) the humanized light chaincomprising three complementarity determining regions, CDR1, CDR2 andCDR3, of the mouse M291 immunoglobulin light chain, and a variableregion framework from a human kappa light chain variable regionframework sequence, and (2) the humanized heavy chain comprising threecomplementarity determining regions, CDR1, CDR2 and CDR3, of the mouseM291 immunoglobulin heavy chain, and a variable region framework from ahuman heavy chain variable region framework sequence provided that atleast one position selected from a group consisting of H30, H67, H68,H70, H72 and H74 is occupied by the same amino acid present in theequivalent position of the mouse M291 immunoglobulin heavy chainvariable region framework; wherein the immunoglobulin specifically bindsto a CD3 antigen on the surface of T cells with a binding affinityhaving a lower limit of about 10⁷ M⁻¹ and an upper limit of aboutfive-times the binding affinity of the M291 immunoglobulin wherein themouse antibody has an IgG1 heavy chain with a variable domain designatedSEQ. ID. No. 11 and a kappa light chain with a variable domaindesignated SEQ. ID. No.
 9. 16. The humanized antibody of claim 15,whereinthe humanized light chain variable region framework is from thelight chain variable region framework of the HF2-1/17 antibody; thehumanized heavy chain region framework is from the heavy chain regionvariable framework of the 21/28 antibody provided that at least oneposition selected from the group is occupied by the same amino acidpresent in the equivalent position of the mouse M291 immunoglobulinheavy chain variable region framework, and provided that position 44 isoccupied by the same amino acid present in the equivalent position of ahuman immunoglobulin subgroup I consensus sequence.
 17. The humanizedantibody of claim 16, whereinthe humanized light chain comprises theamino acid sequence of FIG. 5A (upper) (SEQ ID NO. 8) and the humanizedheavy chain comprises the amino acid sequence of FIG. 5B (upper) (SEQ IDNO. 10).
 18. The bispecific antibody of claim 1 that is humanized. 19.The bispecific antibody of claim 18, wherein the first antigen is theCD3 antigen.
 20. The bispecific antibody of claim 19, comprising:a firstbinding fragment comprising:a humanized form of the heavy chain variableregion of the M291 antibody; a humanized form of the light chainvariable region of the M291 antibody; and a second binding fragment,which is linked to the first binding fragment, comprising:a humanizedform of the heavy chain variable region from the 1D10 antibody; ahumanized form of the light chain variable region from the 1D10antibody; wherein the first binding fragment specifically binds to theCD3 antigen and the second binding fragment specifically binds to the28/32 kDa heterodimeric antigen on the surface of the malignant B cellswherein the mouse M291 antibody has a light chain variable domaindesignated SEQ. ID. No. 9 and a heavy chain variable domain designatedSEQ. ID. No.
 11. 21. The bispecific antibody of claim 20, wherein:thehumanized form of the heavy chain variable region of the M291 antibodycomprises three complementarity determining regions, CDR1, CDR2 andCDR3, of M291 immunoglobulin heavy chain, and a variable regionframework from a human heavy chain variable region framework sequenceprovided that at least one position selected from a group consisting ofH30, H67, H68, H70, H72 and H74 is occupied by the same amino acidpresent in the equivalent position of the mouse M291 immunoglobulinheavy chain variable region framework; the humanized form of the lightchain variable region of the M291 antibody comprises threecomplementarity determining regions, CDR1, CDR2 and CDR3, of the M291immunoglobulin light chain, and a variable region framework from a humankappa light chain variable region framework sequence; the humanized formof the heavy chain variable region. from the 1D10 antibody comprisesthree complementarity determining regions, CDR1, CDR2 and CDR3, of 1D10immunoglobulin heavy chain, and a variable region framework from a humanheavy chain variable region framework sequence provided that at leastone position selected from a second group consisting of H27, H29, H30,H37, H67, H71, H78 and H83 is occupied by the same amino acid present inthe equivalent position of the mouse 1D10 immunoglobulin heavy chainvariable region framework; and the humanized form of the light chainvariable region from the 1D10 antibody comprises three complementaritydetermining regions, CDR1, CDR2 and CDR3, of the 1D10 immunoglobulinlight chain, and a variable region framework from a human kappa lightchain variable region framework sequence provided that at least oneposition selected from a first group consisting of L48, L49, L69, andL70 wherein the amino acid position is occupied by the same amino acidpresent in the equivalent position of the 1D10 immunoglobulin lightchain variable region framework.
 22. The bispecific antibody of claim21, wherein the first binding fragment comprises the heavy chainvariable region shown in FIG. 5B (upper) (SEQ ID NO. 10) and the lightchain variable region shown in FIG. 5A (upper) (SEQ ID NO. 8), and thesecond binding fragment comprises the heavy chain variable region shownin FIG. 4B (upper) (SEQ ID NO. 3) and the light chain variable regionshown in FIG. 4A (upper) (SEQ ID NO. 1).
 23. The bispecific antibody ofclaim 22, wherein the first and second binding fragments each furthercomprises a segment of a constant region fused to the respective heavychain variable regions, and the binding fragments are linked byassociation of the constant regions.
 24. The bispecific antibody ofclaim 23, wherein the binding fragments are Fab or Fab'.
 25. Thebispecific antibody of claim 24, wherein the first and second bindingfragments are Fab's and the bispecific antibody is a F(ab')₂.
 26. Thebispecific antibody of claim 25, wherein the first and second bindingfragments further comprise first and second leucine zippers fused to therespective constant regions.
 27. The bispecific antibody of claim 26,wherein the first binding fragment comprises a heavy chain having theamino acid sequence shown in FIG. 5D (SEQ ID NO. 13) and the secondbinding fragment comprises a heavy chain having the amino acid sequenceshown in FIG. 4D (SEQ ID NO. 6).
 28. A composition comprising thebispecific antibody of claim 1 and a pharmaceutically acceptablecarrier.